MDS_DGE_HHT
Monica Ransom
9/7/2021
This analysis is to look at the MDS samples that were treated with the drug HTT homoharringtonine which is a translation inhibitor. There were only 2 samples for each treatment in this arm.
Pre-processing
- Adapters were trimmed using cutadapt v1.16
- Gene expression was quantified using salmon v1.3.0
- TPMs were obtained for the genes using tximport 1.20.0
library(dplyr)
library(ggplot2)
library(DESeq2)
library(tximport)
library(readr)
library(tximportData)
library(readxl)
library(knitr)
library(tidyverse)
library(pheatmap)
library(RColorBrewer)
library(viridis)
library(ggrepel)
library(EnhancedVolcano)
library(fgsea)
library(limma)
library(VennDiagram)
library(UpSetR)
library(wesanderson)
library(kableExtra)Summary of Data Metrics
| Sample | patient | reads | %Q30 | Duplication rate | % reads with adapter | STAR alignment number | percent aligned | splices annotated | salmon mapping |
|---|---|---|---|---|---|---|---|---|---|
| CD123+ | HTB336 | 28161997 | 93 | 40 | 2.7 | 24795059 | 88.04 | 8802685 | 82.8170 |
| CD123+ | HTB61 | 17643949 | 90 | 29 | 2.5 | 13932164 | 78.96 | 3351481 | 68.1966 |
| CD123+ HHT | HTB336 | 17398408 | 93 | 42 | 2.7 | 15452341 | 88.81 | 5925372 | 87.9806 |
| CD123+ HHT | HTB61 | 22970807 | 93 | 33 | 2.6 | 20607839 | 89.71 | 4897461 | 75.8607 |
Summary of Data prior to analysis
| SampleName | patient | cellType | percentrRNA |
|---|---|---|---|
| HTB61_CD123 | HTB61 | 123pos | 0.30 |
| HTB61_CD132_HHT | HTB61 | 123pos_HHT | 0.14 |
| HTB336_CD123_pos | HTB336 | 123pos | 0.38 |
| HTB336_CD123_pos_HHT | HTB336 | 123pos_HHT | 0.17 |
There are 5001 genes with more than 5 counts in all samples
Sample Heatmap and Correlation matrix
PCA plot
PC1 vs PC2
We see a stronger correlation between patient than between sample type. One principle component is seperating on patient while the other is separating based on sample type.
Run Differential Expression testing using DESeq2 and Calculate Gene Set Enrichment
Compare 123pos vs 123neg, 123neg vs bulk, and 123pos vs bulk
sig = padj <0.01 and abs(l2fc) >0.5
| external_gene_name | padj | pvalue | log2FoldChange |
|---|---|---|---|
| FOS | 3.44e-16 | 2.40e-20 | -4.45 |
| LRPAP1 | 4.77e-14 | 6.65e-18 | -4.62 |
| TM4SF1 | 7.62e-11 | 1.59e-14 | 4.26 |
| OSM | 3.73e-10 | 1.30e-13 | -5.03 |
| HBA2 | 3.73e-10 | 1.26e-13 | -3.61 |
| ICAM1 | 2.69e-09 | 1.13e-12 | -2.96 |
| CD83 | 6.11e-09 | 2.98e-12 | -2.88 |
| HBA1 | 1.86e-07 | 1.04e-10 | -5.37 |
| WASH5P | 5.59e-07 | 3.51e-10 | -5.12 |
| ZNF14 | 5.77e-07 | 4.02e-10 | -2.74 |
| GSDME | 1.52e-06 | 1.16e-09 | -6.11 |
| RPS15 | 3.50e-06 | 3.17e-09 | -2.74 |
| ETV3 | 3.50e-06 | 3.18e-09 | -2.40 |
| SERTAD1 | 3.71e-06 | 3.62e-09 | -3.58 |
| ZNF133 | 5.18e-06 | 5.42e-09 | -5.74 |
| ZNF256 | 1.81e-05 | 2.02e-08 | -4.74 |
| IGLC2 | 1.98e-05 | 2.62e-08 | 6.19 |
| TRAF6 | 1.98e-05 | 2.39e-08 | -2.98 |
| GADD45B | 1.98e-05 | 2.51e-08 | -3.84 |
| ATP2A1 | 2.50e-05 | 3.48e-08 | 4.94 |
| RGS1 | 3.13e-05 | 4.58e-08 | -2.61 |
| SNHG17 | 4.09e-05 | 6.27e-08 | -2.44 |
| UCP2 | 4.69e-05 | 7.53e-08 | -2.56 |
| DPH6 | 5.47e-05 | 9.15e-08 | -3.96 |
| RASGEF1B | 6.50e-05 | 1.18e-07 | -2.09 |
| SIKE1 | 6.50e-05 | 1.14e-07 | -2.65 |
| SLC25A6 | 7.98e-05 | 1.56e-07 | -2.79 |
| ZNF625-ZNF20 | 7.98e-05 | 1.52e-07 | -3.09 |
| ANXA2 | 9.48e-05 | 1.92e-07 | 2.04 |
| CHMP1B-AS1 | 9.51e-05 | 1.99e-07 | -4.70 |
| NT5DC1 | 1.11e-04 | 2.40e-07 | -3.44 |
| PDE4B | 1.16e-04 | 2.68e-07 | -2.13 |
| BSG | 1.16e-04 | 2.67e-07 | -2.67 |
| PHYH | 1.20e-04 | 2.91e-07 | -3.23 |
| ZNF442 | 1.20e-04 | 2.93e-07 | -2.67 |
| CFAP20 | 1.60e-04 | 4.02e-07 | -2.53 |
| MCTP2 | 1.68e-04 | 4.53e-07 | -2.63 |
| C19orf48 | 1.68e-04 | 4.57e-07 | -2.40 |
| PUF60 | 1.68e-04 | 4.37e-07 | -2.85 |
| GSK3B | 1.71e-04 | 4.78e-07 | 3.60 |
| NUDT3 | 1.92e-04 | 5.50e-07 | 2.51 |
| NDUFS7 | 2.47e-04 | 7.24e-07 | -3.52 |
| PRMT9 | 2.69e-04 | 8.07e-07 | -3.32 |
| ATF3 | 2.87e-04 | 8.80e-07 | -2.68 |
| ESS2 | 3.10e-04 | 9.72e-07 | -4.32 |
| NUFIP1 | 3.21e-04 | 1.03e-06 | -4.80 |
| KLF10 | 4.05e-04 | 1.33e-06 | -2.21 |
| PLAUR | 5.10e-04 | 1.71e-06 | -2.09 |
| TNFAIP3 | 5.67e-04 | 1.94e-06 | -2.40 |
| IGHM | 5.75e-04 | 2.05e-06 | -2.91 |
| MUC12 | 5.75e-04 | 2.03e-06 | -5.51 |
| NLRP3 | 6.16e-04 | 2.27e-06 | -2.49 |
| IL1RL1 | 6.16e-04 | 2.28e-06 | -3.85 |
| ABHD10 | 6.52e-04 | 2.46e-06 | -3.98 |
| ZNF140 | 6.59e-04 | 2.53e-06 | -2.21 |
| ARL4A | 6.79e-04 | 2.70e-06 | -1.94 |
| CXCR2 | 6.79e-04 | 2.67e-06 | -9.28 |
| NLE1 | 6.99e-04 | 2.83e-06 | -4.05 |
| NCKAP1 | 1.05e-03 | 4.34e-06 | 3.59 |
| PPP1R15A | 1.08e-03 | 4.51e-06 | -2.76 |
| LTBP1 | 1.13e-03 | 4.80e-06 | -7.56 |
| NANS | 1.14e-03 | 4.92e-06 | -2.19 |
| WDR43 | 1.26e-03 | 5.53e-06 | -1.99 |
| CDKN1A | 1.27e-03 | 5.68e-06 | -2.04 |
| NFU1 | 1.27e-03 | 5.76e-06 | -3.16 |
| CIDEB | 1.27e-03 | 5.90e-06 | 6.16 |
| RPL8 | 1.27e-03 | 5.94e-06 | -1.77 |
| HAUS7 | 1.30e-03 | 6.24e-06 | -3.36 |
| HEMK1 | 1.30e-03 | 6.26e-06 | -2.35 |
| FFAR3 | 1.45e-03 | 7.18e-06 | -8.78 |
| ZNF394 | 1.45e-03 | 7.10e-06 | -2.08 |
| BTBD8 | 1.53e-03 | 7.70e-06 | -8.97 |
| MTX2 | 1.54e-03 | 7.83e-06 | -4.46 |
| GRAP | 1.55e-03 | 7.98e-06 | -9.19 |
| IFFO1 | 1.72e-03 | 8.98e-06 | -8.07 |
| TNFAIP6 | 1.72e-03 | 9.11e-06 | -2.49 |
| PLA2G12A | 1.75e-03 | 9.41e-06 | -3.71 |
| CLEC4E | 1.80e-03 | 9.80e-06 | -5.24 |
| RNF138 | 1.85e-03 | 1.02e-05 | -2.14 |
| PTRHD1 | 1.85e-03 | 1.03e-05 | -3.55 |
| HNRNPDL | 1.87e-03 | 1.06e-05 | -1.87 |
| RALY | 1.92e-03 | 1.10e-05 | 3.82 |
| IVD | 1.95e-03 | 1.13e-05 | -2.50 |
| R3HCC1 | 2.18e-03 | 1.28e-05 | -3.98 |
| PMAIP1 | 2.91e-03 | 1.73e-05 | -2.40 |
| H1-4 | 2.91e-03 | 1.75e-05 | -3.24 |
| FOSB | 3.00e-03 | 1.82e-05 | -2.03 |
| WDFY2 | 3.06e-03 | 1.90e-05 | 2.33 |
| ARID4A | 3.06e-03 | 1.88e-05 | -1.90 |
| NKTR | 3.07e-03 | 1.93e-05 | -1.91 |
| EIF1 | 3.46e-03 | 2.19e-05 | -1.97 |
| ASH2L | 3.59e-03 | 2.30e-05 | -2.17 |
| USP3 | 3.71e-03 | 2.41e-05 | -2.25 |
| SNX17 | 4.02e-03 | 2.64e-05 | -2.79 |
| CYB5R4 | 4.22e-03 | 2.80e-05 | -2.17 |
| POLR2E | 4.28e-03 | 2.89e-05 | -3.21 |
| BTG2 | 4.28e-03 | 2.89e-05 | -2.40 |
| RNF144B | 4.38e-03 | 3.02e-05 | -2.40 |
| LMAN2 | 4.38e-03 | 3.00e-05 | -2.78 |
| CXCR5 | 4.38e-03 | 3.08e-05 | -9.00 |
| IRAK2 | 4.38e-03 | 3.08e-05 | -2.81 |
| H2AC19 | 4.41e-03 | 3.14e-05 | -5.21 |
| USP11 | 4.52e-03 | 3.25e-05 | -2.49 |
| GFI1B | 4.53e-03 | 3.32e-05 | -2.86 |
| ACSF3 | 4.53e-03 | 3.30e-05 | -3.43 |
| DOCK7 | 4.54e-03 | 3.40e-05 | 3.45 |
| SLC7A6OS | 4.54e-03 | 3.36e-05 | -3.84 |
| HSPA5 | 4.54e-03 | 3.42e-05 | -1.82 |
| JDP2 | 5.26e-03 | 4.00e-05 | 4.12 |
| 5.35e-03 | 4.10e-05 | -8.86 | |
| ZBTB48 | 5.53e-03 | 4.28e-05 | -2.76 |
| RGS2 | 6.13e-03 | 4.83e-05 | -2.06 |
| 6.13e-03 | 4.82e-05 | 4.33 | |
| ZNF136 | 6.19e-03 | 4.92e-05 | -1.96 |
| SAMSN1 | 6.33e-03 | 5.12e-05 | -1.74 |
| ZFP36 | 6.33e-03 | 5.16e-05 | -2.29 |
| SPPL2A | 6.33e-03 | 5.09e-05 | 2.38 |
| SRSF10 | 6.50e-03 | 5.34e-05 | -1.67 |
| POC5 | 6.79e-03 | 5.68e-05 | -3.00 |
| SNAPC1 | 6.79e-03 | 5.68e-05 | -2.08 |
| HLA-E | 7.10e-03 | 5.99e-05 | -1.62 |
| CDC37L1 | 7.17e-03 | 6.10e-05 | -2.62 |
| SDE2 | 7.40e-03 | 6.35e-05 | -1.76 |
| MBNL3 | 7.42e-03 | 6.42e-05 | -4.55 |
| DNAJB1 | 8.24e-03 | 7.18e-05 | -1.82 |
| FLRT2 | 8.45e-03 | 7.42e-05 | 3.48 |
| MIR222HG | 8.68e-03 | 7.72e-05 | 2.52 |
| CISH | 8.68e-03 | 7.75e-05 | -3.55 |
| HIF1AN | 9.24e-03 | 8.31e-05 | -2.14 |
| UGGT2 | 9.36e-03 | 8.49e-05 | -4.59 |
| DDA1 | 9.43e-03 | 8.61e-05 | -3.39 |
| LIPN | 1.00e-02 | 9.20e-05 | -8.60 |
Volcano Plot
###MA plots 
##Displaying a table of ordered pathways
#plot the waterfall results
All Pathways In Moustache Plot
##repeating with KEGG pathways
Top Up and Down Ranked Pathways hallmark
Top Up and Down Ranked Pathways kegg
Top ranked pathway hallmark
I can do this for any of interest, just let me know 
Top ranked pathway kegg
I can do this for any of interest, just let me know 